Capillary isoelectric focusing of haemoglobin variants in the clinical laboratory

Abstract

For capillary isoelectric focusing (CIEF) to be accepted in the clinical laboratory, it must be reproducible and cost effective. The advent of polyAAEE coated capillaries (Bio-Rad Laboratories, Hercules, CA, USA) has provided the means of obtaining over 100 runs per capillary, something which previously had not always been possible with coated capillaries. Using the Clinical Data Management computer program on the BioFocus 2000 Capillary Electrophoresis System (Bio-Rad Laboratories), we have used a one-step salt mobilization to achieve focusing of haemoglobin variants. Washed red cells are diluted, haemolyzed and separated in the capillary at 8 kV using 1.3% Pharmalyte ampholytes (pH 6.6–7.7/pH 6–8 2:1) in 0.40% methylcellulose. The separated haemoglobins were detected by adsorption at 280 nm. Using published values of haemoglobin variants, we investigated the use of pI markers to confirm the pI of haemoglobin variants detected. CIEF, though more expensive than capillary electrophoretic separations of haemoglobin variants, has greater resolution due to the fact that the separation of variants from pI 6.95 to 7.42 occurs over 4 min, whereas the electrophoretic separation is over 60 s. CIEF is quicker than gel IEF, and shows real-time results as the sample is being processed. The potential for CIEF in the clinical laboratory is not limited to haemoglobin variants, and the technique should become increasingly popular in the near future.